This application entitled "Attenuation of Diverse Cell Death Pathways after Traumatic Brain Injury by Multi- drug Combination Therapy" addresses Broad Challenge Area (15): Translational Science and Specific Challenge Topic: 15-HD-104, Multi-drug Combination Therapy for TBI and Stroke Treatment. The purpose of the proposed studies is to evaluate a novel combination treatment strategy in experimental traumatic brain injury (TBI). Traditional neuroprotective treatment strategies for TBI aim to prevent delayed (secondary) neuronal cell death, generally by inhibiting one proposed cell death mechanism. Yet considerable research indicates that multiple pathways and mechanisms of cell death contribute to tissue loss. In focal TBI, the central injury site is thought to largely reflect necrotic cell death, which primarily occurs within the first 6-8 hours after trauma and is associated with severe bioenergetic compromise. Our recent work, however, indicates that in several rodent TBI models -mouse controlled cortical impact (CCI) or rat lateral fluid percussion (LFP)- cell death in the central core region also includes a substantial component of caspase-independent apoptosis, whereas the better known caspase-dependent cell death is detected in the more peripheral regions where ATP and ADP levels are largely preserved. The caspase-independent programmed cell death (PCD) is due in large part to the activation/translocation of apoptosis-inducing factor (AIF). Such cell death occurs relatively late after injury (24-72h) and can be inhibited by delayed treatment hours after the insult. These data raise serious questions about classical assumptions regarding mechanism of post-traumatic brain injury, suggesting the possibility of newer treatment approaches with extended therapeutic windows and explaining why, if used exclusively, caspase inhibitors may have only a partial protective effect. Ideally, treatment for TBI should attempt to inhibit both caspase-independent and caspase-dependent PCD. Recent studies in ischemia, as well as unpublished work from our laboratory, suggest two intriguing approaches for limiting the three major pathways of cell death after injury. One approach is to up-regulate heat shock protein 70 (HSP70), which binds both Apaf-1 and AIF at distinct sites, thereby neutralizing their pro- apoptotic functions by preventing the formation of the apoptosome (and caspase 3) and attenuating AIF mediated actions. Our preliminary data show that TBI causes up-regulation of HSP70 in many neurons within the injury zone; those neurons expressing HSP 70 show neither caspase-3 activation nor AIF translocation. Up-regulation of HSP70 in cerebral ischemia is strongly protective. The other approach is to inhibit PARP-1. PARP-1, through the release of poly ADP ribose (PAR), is a critical upstream activator AIF release from neuronal mitochondria; it also more recently has been shown to be a critical activator of microglia. Inhibition of PARP-1 after TBI strongly attenuates both caspase dependent and independent forms of PCD, as well as microglial activation, with markedly improved outcome. The advantage of each of these strategies is that their therapeutic window should be very broad, at least 24h. By combining these distinct therapeutic strategies, additive or synergistic protective effects may potentially be achieved. Should our hypotheses be supported, concepts regarding treatment of TBI will be markedly altered and target populations for therapeutic intervention considerably expanded. Combination treatment evaluation is proposed in two pathobiologically different models in different species, with the assumption that potent treatment effects duplicated across models and species makes ultimate clinical translation more likely. Specific hypotheses include: 1) HSP-70 inducers or PARP-1 inhibitors attenuate caspase-independent and caspase-dependent PCD after TBI, reducing long-term neurological dysfunction; 2) Each treatment approach has a long therapeutic window of at least 24h; 3) combined therapy with HSP-70 inducers and PARP-1 inhibitors demonstrate additive and/or synergistic effects in both mouse CCI and rat LFP models. We propose the following specific aims: 1) to compare the efficacy, dose response, and therapeutic window of two structurally distinct HSP-70 inducers with regard to attenuation of post-traumatic neuronal cell death and improved functional recovery after moderate CCI injury in mice; 2) to compare the efficacy, dose response and therapeutic window of two structurally distinct PARP-1 inhibitors with regard to attenuation of post-traumatic neuronal cell death and improved functional recovery after moderate CCI injury in mice; 3) to determine whether combined multi-drug therapy with the best HSP-70 inducer and best PARP-1 inhibitor, at optimal doses, has additive or synergistic effects on cell death, microglial activation, and neurodegenerative conditions up to 3 months post-injury. The Centers for Disease Control and Prevention (CDC) defines traumatic brain injury (TBI) as craniocerebral trauma associated with a decreased level of consciousness, amnesia, other neurologic or neuropsychological abnormalities, skull fracture, intracranial lesions, or death. It has been reported that the combined incidence of fatal and hospitalized TBI among all age groups has a median annual incidence of 101 per 100,000. Approximately 20% of TBIs cause death either immediately or during acute hospital care, with estimated annual rates of mild TBI treated only in outpatient facilities or hospital emergency departments (EDs) in the United States were 392 and 540 visits per 100,000, respectively. A disability prevalence of 37% has been reported for TBI patients followed more than one year after hospitalization; based on this figure, the CDC has estimated that nearly 2% of the entire US population has TBI-related disabilities. Traumatic brain injury (TBI) may occur in as many as 22% of troops deployed in Afghanistan and Iraq. Recent studies in ischemia, as well as unpublished work from our laboratory, suggest two intriguing approaches for limiting the three major pathways of cell death after injury. One approach is to up-regulate heat shock protein 70 (HSP70), which binds both Apaf-1 and AIF at distinct sites, thereby neutralizing their pro-apoptotic functions by preventing the formation of the apoptosome (and caspase 3) and attenuating AIF mediated actions. Our preliminary data show that TBI causes up- regulation of HSP70 in many neurons within the injury zone; those neurons expressing HSP 70 show neither caspase-3 activation nor AIF translocation. Up-regulation of HSP70 in cerebral ischemia is strongly protective. The other approach is to inhibit PARP-1. PARP-1, through the release of poly ADP ribose (PAR), is a critical upstream activator AIF release from neuronal mitochondria; it also more recently has been shown to be a critical activator of microglia. Inhibition of PARP-1 after TBI strongly attenuates both caspase dependent and independent forms of PCD, as well as microglial activation, with markedly improved outcome. The advantage of each of these strategies is that their therapeutic window should be very broad, at least 24h. By combining these distinct therapeutic strategies, additive or synergistic protective effects may potentially be achieved. Should our hypotheses be supported, concepts regarding treatment of TBI will be markedly altered and target populations for therapeutic intervention considerably expanded. Combination treatment evaluation is proposed in two pathobiologically different models in different species, with the assumption that potent treatment effects duplicated across models and species makes ultimate clinical translation more likely.